U.S. patent number 5,750,472 [Application Number 08/367,130] was granted by the patent office on 1998-05-12 for laminarin as a seed germination and plant growth accelerator.
This patent grant is currently assigned to Laboratoires Goemar S.A.. Invention is credited to Cyrille Amin-Gendy, Bernard Cloarec, Florence Levasseur, Yvette Janine Lienart, Pascale Patier, Cyrille Rochas, Kiem-Ngoe Tran Thanh, Jean-Claude Yvin.
United States Patent |
5,750,472 |
Yvin , et al. |
May 12, 1998 |
Laminarin as a seed germination and plant growth accelerator
Abstract
The present invention relates to the use of laminarin as a seed
germination and plant growth accelerator. The invention applies to
the field of agriculture for treatments via the leaves or to the
soil.
Inventors: |
Yvin; Jean-Claude (Saint Malo,
FR), Levasseur; Florence (Saint Malo, FR),
Amin-Gendy; Cyrille (Argenteuil, FR), Tran Thanh;
Kiem-Ngoe (Gif sur Yvette, FR), Patier; Pascale
(Tassin-la-Deth-Lune, FR), Rochas; Cyrille (La Combe
de Lancey, FR), Lienart; Yvette Janine (St Nizier
d'Uriage, FR), Cloarec; Bernard (Saint Pol de Leon,
FR) |
Assignee: |
Laboratoires Goemar S.A. (Saint
Malo, FR)
|
Family
ID: |
9431643 |
Appl.
No.: |
08/367,130 |
Filed: |
March 14, 1995 |
PCT
Filed: |
July 06, 1993 |
PCT No.: |
PCT/FR93/00698 |
371
Date: |
March 14, 1995 |
102(e)
Date: |
March 14, 1995 |
PCT
Pub. No.: |
WO94/00993 |
PCT
Pub. Date: |
January 20, 1994 |
Foreign Application Priority Data
|
|
|
|
|
Jul 7, 1992 [FR] |
|
|
92 08387 |
|
Current U.S.
Class: |
504/292 |
Current CPC
Class: |
A01N
43/16 (20130101); A01N 65/00 (20130101); A01N
65/08 (20130101); A01N 65/10 (20130101); A01N
65/12 (20130101); A01N 65/20 (20130101); A01N
65/34 (20130101); C05F 11/00 (20130101); A01N
65/38 (20130101); A01N 65/40 (20130101); A01N
65/44 (20130101); A01N 65/00 (20130101); A01N
2300/00 (20130101) |
Current International
Class: |
A01N
43/16 (20060101); A01N 43/02 (20060101); A01N
65/00 (20060101); C05F 11/00 (20060101); A01N
043/16 () |
Field of
Search: |
;504/292 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 218 770 |
|
Apr 1987 |
|
EP |
|
0 538 091 |
|
Apr 1993 |
|
EP |
|
3 735 365 |
|
Apr 1988 |
|
DE |
|
58 144 309 |
|
Aug 1983 |
|
JP |
|
62-265 208 |
|
Nov 1987 |
|
JP |
|
3 034 905 |
|
Feb 1991 |
|
JP |
|
Other References
"Beta-(1,3) Glucanase Activity and Quantity of Fungus in Relation
to Fusarium Wilt in Resistant and Susceptible Near-Isogenic Lines
of Muskmelon", by D. Netzer et al, Physiol Plant Pathol, vol. 14,
No. 1, 1979, pp. 47-56.(Biological Abstracts, vol. 68, No. 1, 1979,
abstract No. 5199). .
"Induction, Purification and Possible Function of Chitinase in
Cultured Carrot Cells", by F. Kurosaki et al, Physiol Mol Plant
Pathol, vol. 31, No. 2, 1987, pp. 201-210. (Biological Abstracts,
vol. 85, No. 2, Jan. 15, 1988, abstract No. 20313). .
"Elicitor-Induced Accumulation of Glyceollin and Callose in Soybean
Roots and Localized Resistance Against Phytophora Megasperma F. SP.
Glycinea", by A. Bonhoff et al, Plant Sci, vol. 54, No. 3, 1988,
pp. 203-210. (Biological Abstracts, vol. 86, No. 4, 1998, abstract
No. 41880). .
"Chitin and Related Compounds as Elicitors of the Lignification
Response in Wounded Wheat", by R.B. Pearce et al, Physiol Plant
Pathol, vol. 20, No. 1, 1982, pp. 119-124. (Biological Abstracts,
vol. 74, No. 8, 1982, abstract No. 56641). .
"Enzymic Degradation of the Endosperm Cell Walls of Germinated
Sorghum", by G.H. Palmer, World J. Microbiol Biotechnol, vol. 7,
No. 1, 1991, pp. 17-21. (Biological abstracts, vol. 91, abstract
No. 123505). .
"Carbohydrate-Degrading Enzymes in Germinating Wheat", by A.M.
Corder et al, Cereal Chem, vol. 66, No. 5, 1989, pp. 435-439.
(Biological Abstracts, vol. 89, abstract No. 510384). .
"Effect of Carbohydrates in Seaweed Fertilizers", by G. Blunden et
al, Proc. Int. Seaweed Symp., 6th Meeting Date 1968; 1969, pp.
647-653. (Chemical Abstracts, vol. 74, No. 5, abstract No. 22224f).
.
"Induction and Secretion of Alpha Amylase 1-3 1-4-Beta Glucanase
and 1-3-Beta Glucanase Activities in Gibberellic Acid and Calcium
Dichloride-Treated Half Seeds and Aleurones of Wheat", by A.M.
Bernier et al, Cereal Chem, vol. 70, No. 2, 1993, pp. 127-132.
(Biological Abstracts, vol. 95, abstract No. 115659). .
"Manufacture of Algal Chemicals. III. Laboratory-Scale Isolation of
Laminarin from Brown Marine Algae", Black et al., J. appl. Chem.,
I, Nov. 1951, pp. 505-517. .
"Pharmacological Activities of Marine Algae", Guven et al.,
Introduction to Applied Phycology, Editor I. Akatsuka, SPB Academic
Publishing, 1990, pp. 67-92. .
Bonhoff et al, "Elicitor-Induced Accumulation of Glyceollin . . . "
Plant Science 54:203-209. 1988. .
Bernier et al, "Induction and Secretion of .alpha.-Amylase . . . "
Cereal Chemistry 70(2):127-132. Feb. 1993..
|
Primary Examiner: Clardy; S. Mark
Attorney, Agent or Firm: Lowe, Price, LeBlanc &
Becker
Claims
We claim:
1. A method for accelerating a plant's growth comprising a step of
applying a composition containing an effective amount of laminarin
to the leaves of said plant.
2. A method for accelerating the growth of a plant comprising a
preliminary step of determining the effective amount of laminarin
that will accelerate the growth of said plant and a further step of
applying a composition containing said effective amount of
laminarin to the leaves of said plant.
3. The method as defined in claim 2, wherein said composition
containing an effective amount of laminarin is in liquid form.
4. The method as defined in claim 3, wherein said liquid
composition containing an effective amount of laminarin contains
0.005 to 10% by weight of laminarin.
5. The method as defined in claim 3, wherein said composition
containing an effective amount of laminarin also includes at least
one additional substance selected from the group consisting of
deficiency-correcting elements, fungicides, insecticides,
herbicides, growth hormones, lipoamino acids and betaines.
6. A method for accelerating seed germination comprising a step of
applying a composition containing an effective amount of laminarin
to a seed.
7. The method as defined in claim 6, wherein said composition
containing an effective amount of laminarin is applied to the seed
by being added to the soil in which the seed is planted.
8. The method as defined in claim 7, wherein said composition
containing an effective amount of laminarin is in solid form.
9. The method as defined in claim 7, wherein said composition
containing an effective amount of laminarin is in liquid form.
10. The method as defined in claim 7, wherein said effective amount
of laminarin is in the range of 1 g to 100 g/100 kg of seeds.
11. The method as defined in claim 7, comprising a preliminary step
of determining the effective amount of laminarin that will
accelerate the germination of the seed to which said composition is
applied.
12. The method as defined in claim 6, wherein said composition
containing an effective amount of laminarin is in solid form.
13. The method according to claim 12, wherein said composition
contains from 0.005 to 50% by weight of laminarin.
14. The method as defined in claim 6, wherein said composition
containing an effective amount of laminarin is in liquid form.
15. The method as defined in claim 14, wherein said composition
containing an effective amount of laminarin is in the form of a
film coating solution.
16. The method according to claim 14, wherein said composition
contains from 0.005 to 50% by weight of laminarin.
17. The method as defined in claim 6, wherein said composition
containing an effective amount of laminarin includes at least one
additional substance selected from the group consisting of
deficiency-correcting elements, fungicides, insecticides,
herbicides, growth hormones, lipoamino acids and betaines.
18. The method as defined in claim 6, wherein said effective amount
of laminarin in the range of 1 g to 100 g/100 kg of seeds.
19. The method as defined in claim 6, comprising a preliminary step
of determining the effective amount of laminarin that will
accelerate the germination of the seed to which said composition is
applied.
Description
This application has been filed under 35 USC 371 from international
application PCT/FR93/00698, filed Jul. 6, 1993.
The present invention relates to the use of laminarin as a seed
germination and plant growth accelerator.
It is known that laminarin is a storage polymer of brown algae and
consists of polysaccharides whose structures differ slightly
according to the nature of the alga.
In general terms, laminarin consists of 20 to 60 D-glucopyranoside
units distributed in a main linear chain, in which these units are
joined by .beta.(1-3) linkages, and branches joined to this main
chain by .beta.(1-6) linkages.
Some of these chains have a reducing terminal unit consisting of a
mannitol unit. The existence of mannose units within this structure
is also noted.
Laminarin is generally extracted from brown algae of the
Pheophyceae type, and in particular the Fucales or the
Laminariales.
Various extraction methods can be used to obtain the laminarin.
Reference may be made for example to the method described by Black
et al., Appl. Chem. (1951), volume 1, pages 505 to 517.
More generally, laminarin can be obtained from brown algae by any
extraction process which enables the constituents other than
laminarin (wall polysaccharides, salts, etc.) to be successively
removed.
In particular, these processes use steps involving grinding,
precipitation in an acid or basic medium, ultrafiltration and
dialysis.
Laminarin is also marketed, for example by Sigma Chimie SARL.
It is further known that sulfated laminarin possesses valuable
pharmacological properties, in particular anticoagulant and
hypocholesterolemic properties (K. C. Guven et al., Introduction to
Applied Physiology, 1990, pages 67 to 92).
Various scientific publications describe the eliciting properties
of laminarin, suggesting its use for enhancing plant defense
reactions.
Thus NETZER et al. reveal that an infection with the pathogen S.
oxysporum triggers the induction of laminarinase (Biological
abstracts, vol. 68, no. 1, 1979).
Likewise, BONHOFF et al. reveal the properties of laminarin as an
elicitor of phytoalexin and callose (Biological abstracts, vol. 86,
no. 4, 1988).
Furthermore, KUROSAKI et al. (Biological abstracts, vol. 85, no. 2,
1988) and PEARCE (Biological abstracts, vol. 74, no. 8, 1982)
confirm the eliciting effects of laminarin, in particular in
respect of lignification, while at the same time stating that these
effects are weak compared with those of the known elicitors.
This state of the art might suggest to those skilled in the art
that they use laminarin to enhance plant defense reactions.
It has been discovered, and this is the basis of the invention,
that laminarin has the properties of an alpha-amylase elicitor,
causing an accelerating action on seed germination and plant
growth.
It is pointed out that elicitation is a process which is based on a
phase in which a metabolite is recognized by the plant cell and a
phase in which a physiological response is generated by a signal.
The elicitor therefore corresponds to any signal capable of
inducing in plants reactions which enable it to adapt to its
environment.
It has been discovered, quite unexpectedly, that laminarin induces
the activation of marker enzymes of the growth process in plants,
and the stimulation of the proteolytic activity of the treated
cells.
It has thus been demonstrated that laminarin and compositions in
which it is present increase the production yield of agricultural
products by exerting an accelerating action on plant growth, cell
elongation and seed germination.
Thus the present invention relates to the use of laminarin as a
seed germination and plant growth accelerator.
According to one particular characteristic of this use according to
the invention, the laminarin is applied via the leaves or to the
soil.
According to another particular characteristic of the invention,
this use comprises the preparation of a composition containing an
effective amount of laminarin, optionally incorporated with an
agriculturally acceptable carrier or vehicle.
According to one particular characteristic, this composition is
presented in solid form, especially in the form of powders or
granules, or in liquid form, especially in the form of aqueous
solutions.
According to another particular characteristic, this composition
also contains at least one additional substance selected from
deficiency-correcting elements, fungicides, insecticides,
herbicides, growth hormones, lipoamino acids and betaines.
Examples of herbicides which can be used are compounds belonging to
the group comprising carbamates, cyclohexadiones, sulfonylureas,
triazines or uracils.
Examples of fungicides which can be used are compounds belonging to
the group comprising dithiocarbamates, hydantoins, imidazoles or
triazoles.
Advantageously, this composition contains from 0.005 to 503 by
weight of laminarin and preferably from 1 to 253 by weight when it
is in solid form, and from 0.005 to 103 by weight of laminarin and
preferably from 0.5 to 53 by weight when it is in liquid form.
In general terms, the compositions consistent with the use
according to the invention are prepared by mixing laminarin,
extracted in powder form, with customary additives, for example
solid fillers and/or solvents. Surface-active substances,
dispersants or emulsifiers will also be used.
Examples of solid fillers which can be used for the preparation of
wettable powders or granules are finely divided kaolin or clay.
When presented in liquid form, these compositions will preferably
be obtained by diluting the laminarin in water.
It is also possible to envisage the preparation of spraying
compositions based on oil or emulsifiable concentrate, especially
in the case of a mixture with additional substances which are
insoluble in water.
Examples of plants which can be treated successfully with laminarin
within the framework of the use according to the invention are:
fruit crops such as apple trees, pear trees and vines;
cereals such as wheat, maize and rice;
oleaginous plants such as soya, sunflower and colza;
vegetables such as carrots, tomatoes, cauliflowers and
potatoes.
In general, laminarin must be used in doses of between 0.005 g and
100 g per liter for treating the leaves, and of between 1 g and 100
g per 100 kg for treating the seeds.
BRIEF DESCRIPTION OF THE DRAWING
The drawing graphically represents the results obtained by
enzymatic activation (R) defined as the ratio of the activity
measured in elicited cells or protoplasts to the activity measured
in untreated cells or protoplasts. E1 and E2 designate the
elicitors tested. Each value plotted on the graph results from the
processing of pre-experimental data obtained in the course of an
elicitation experiment. E1 induces an alpha-amylase activation R of
300% in the cells, whereas laminarin-based composition E2 induces
an alpha-amylase activation R of 600%.
The present invention will be illustrated with the aid of the
following non-limiting Examples:
First of all, Examples 1 and 2 will give two processes for the
extraction of laminarin from a brown alga.
The NMR spectra of the extracts obtained by carrying out the
processes described in Examples 1 and 2 contain the characteristic
peaks of a commercial laminarin.
EXAMPLE 1
Process for the Extraction of Laminarin from a Brown Alga
300 g of fresh algae of the Laminaria saccharina type, harvested in
August, are subjected to cryocomminution (-40.degree. C.) by the
process described in French patent no. 74 35162.
The product thus obtained has a mean particle diameter of between
50 and 100 .mu.m and a solids content of 10-124%. 0.9 l of 0.34
sulfuric acid is added gradually to 300 g of this product.
Extraction is performed in a water bath at a temperature of about
80.degree. C. for 1 hour, with stirring.
This operation is repeated twice.
After neutralization, the extract obtained is treated with
polyvinylpyrrolidone in a dose of about 14 by weight. This is done
by introducing 9 g of polyvinylpyrrolidone (PVP) into a volume of
90 ml of extract. The PVP is left to thicken for about 2 hours. The
resulting solution is added to about 0.9 l of extract, the mixture
being stirred for 30 min and then filtered under vacuum on a
Whatman GF/A filter.
The liquid obtained in this way is subjected to tangential
ultrafiltration on a carbon-ceramic tubular membrane of the
"Carbosep" type with a porosity of 50,000 Daltons. A pressure of 1
bar is maintained on the filtration column during the
operation.
This gives a filtrate having a volume of about 0.8 liter and a pH
of 5.5. The filtrate is then dialyzed on a cellulose ester membrane
of the SPECTRA/ Por type with a porosity of 500 or 1000 Daltons.
The dialyzate is then lyophilized to give 7 g of dry powder,
corresponding to pure laminarin.
EXAMPLE 2
Alternative Process for the Extraction of Laminarin from a Brown
Alga
300 g of fresh algae of the Ascophyllum nodosum type are ground in
a knife mill of trademark STEFAN, type VCM5. The resulting product
has a particle diameter of less than 1 mm and a solids content of
10-12%. 0.9 l of a 2% aqueous solution of calcium chloride is added
to 300 g of this product, precipitating the alginates in
particular.
The first extraction is performed in a water bath at a temperature
of about 60.degree. C. for 7 hours, with stirring. The first
extract is then recovered by filtration and the 300 g of ground
product are extracted a second time in 0.9 l of a 25 aqueous
solution of calcium chloride under the same conditions as the first
extraction, i.e. in a water bath at a temperature of about
60.degree. C., but this time overnight, with stirring. Then the
second extract is also recovered by filtration.
The two extracts obtained in this way are then combined and
subjected to diafiltration to remove the mineral salts.
This was done using a Pellicon system marketed by Millipore,
equipped with a cassette of porosity 1000 Daltons and associated
with a Procon pump, also marketed by Millipore.
The decrease in the ion content of the extract is followed by
regular measurement of the resistivity.
The diafiltration is continued until the resistivity of the residue
is about 1000 Ohms.cm.
The residue is then treated on a column of anion exchange resin of
the strong exchanger type, such as Amberlite IRA 400, or of the
weak exchanger type, such as AMBERLITE IR 45.
Two techniques can be used:
either a batch process: After regeneration with sodium hydroxide
and rinsing with demineralized water, the resin is placed directly
in a vessel containing the residue. The mixture is stirred for 1
hour and decanted and the filtrate is recovered.
or column chromatography: After regeneration with sodium hydroxide
and rinsing with demineralized water, the resin is introduced into
a glass column of diameter 2-3 cm to a height of 20 cm. The
filtrate is then deposited on the top of the column and caused to
penetrate slowly into the resin. The column is then rinsed with
demineralized water.
The eluate is then dialyzed on a cellulose ester membrane of the
Spectra/Por type with a porosity of 500 or 1000 Daltons. The
dialyzate is then lyophilized to give 7 g of dry powder,
corresponding to pure laminarin.
EXAMPLE 3
Demonstration of the Properties of Laminarin
The properties of laminarin and compositions in which it is present
were demonstrated by performing the following experiments:
Experiment No. 1
Seed Germination Test
The influence of laminarin on seed germination was evaluated using
the following protocol:
Without prior soaking, carrot, lettuce and endive seeds are
germinated in batches of 10 in Petri dishes of diameter 5 cm:
in an aqueous solution (control batch),
in a solution containing growth substances (kinetin or indolacetic
acid (IAA) at concentrations of 10.sup.-6 M), and
in a solution containing laminarin at different concentrations.
The Petri dishes are placed in an enclosure at 25.degree. C. in the
dark.
The results obtained after 10 days are collated in the following
Table:
______________________________________ % GERMINATION PRODUCT TESTED
carrot lettuce endive ______________________________________ Water
35 11 12 IAA 10.sup.-6 M 30 0 15 Kinetin 10.sup.-6 M 27 26 12
Laminarin 0.1 .mu.g/ml 25 16 15 Laminarin 0.2 .mu.g/ml 34 20 13
Laminarin 0.4 .mu.g/ml 47 24 17 Laminarin 0.6 .mu.g/ml 45 34 20
Laminarin 0.8 .mu.g/ml 100 33 10 Laminarin 1 .mu.g/ml 27 32 15
Laminarin 2 .mu.g/ml 41 17 12 Laminarin 4 .mu.g/ml 34 26 9
Laminarin 6 .mu.g/ml 24 30 9 Laminarin 8 .mu.g/ml 35 19 24
Laminarin 10 .mu.g/ml 41 26 13
______________________________________
Therefore, under the experimental conditions tested, laminarin has
an optimal activity at concentrations of:
0.8 .mu.g/ml for carrot seeds,
0.6, 0.8 and 1 .mu.g/ml for lettuce seeds, and
0.4, 0.6 and 0.8 .mu.g/ml for endive seeds.
The results thus demonstrate the influence of laminarin on the
germination of different types of seeds.
Experiment No. 2
Elongation of Wheat Coleoptiles
Wheat seeds are germinated at 25.degree. C. in the dark.
The coleoptiles reach a length of 2.5 to 3 cm after 3 days. After
excision of the coleoptiles, segments 8 mm in length are cut
out.
To determine whether the apical zone, which is the center of
endogenous production of auxin, will interfere synergistically or
antagonistically with the laminarin, two batches were tested, one
containing the apical zone and the other not.
For each experiment, two batches of 10 coleoptile fragments are
brought into contact with 5 ml of an aqueous solution of the test
product (IAA 10.sup.-6 M and laminarin at different concentrations)
in Petri dishes of diameter 5 cm.
The elongation is measured after 20 hours of incubation in the dark
at 25.degree. C.
The results obtained are collated in the following Table:
______________________________________ TREATMENT AVERAGE ELONGATION
______________________________________ Water 9 .+-. 0.87 IAA
10.sup.-6 M 11.12 .+-. 0.29 Laminarin 0.1 .mu.g/ml 12 .+-. 0.33
Laminarin 0.2 .mu.g/ml 12.85 .+-. 0.99 Laminarin 0.4 .mu.g/ml 11
.+-. 0.53 Laminarin 0.6 .mu.g/ml 10.33 .+-. 0.86 Laminarin 0.8
.mu.g/ml 11.83 .+-. 1.54 Laminarin 1 .mu.g/ml 11.16 .+-. 1.67
Laminarin 2 .mu.g/ml 11.16 .+-. 1.67 Laminarin 4 .mu.g/ml 11.25
.+-. 1 Laminarin 6 .mu.g/ml 10.42 .+-. 0.7 Laminarin 8 .mu.g/ml
11.14 .+-. 0.97 Laminarin 10 .mu.g/ml 11.33 .+-. 1.07
______________________________________
Under the experimental conditions tested, laminarin has an optimal
activity at concentrations of 0.1 and 0.2 .mu.g/ml.
Laminarin has a very favorable activity on the elongation of wheat
coleoptiles. It therefore acts as a growth regulator.
EXAMPLE 4
Demonstration of the Properties of Laminarin towards
Alpha-Amylase
The properties towards alpha-amylase of laminarin and compositions
in which it is present were demonstrated by performing the
following experiment:
PROTOCOL
Suspensions of cells or protoplasts of Rubus fructicosus, i.e.
2.4.10.sup.6 cells suspended in 50 ml of sodium citrate buffer, are
incubated for 60 minutes at room temperature, with circular
stirring at 80 rpm, in the presence or absence of elicitors.
The elicitors tested correspond to the laminarin-containing
compositions produced in Examples no. 1 and no. 2, called E1 and E2
respectively.
The concentrations of E1 and E2 used for this elicitation test are
0.1 g of ose per liter.
At the end of the experiment, the cells or protoplasts are
recovered by centrifugation (500 g, 5 minutes at 4.degree. C.) and
taken up in 50 mM Tris-HCl buffer (pH 7.2) enriched in NaCl (1M).
The cells recovered by centrifugation (7300 g, 10 minutes at
4.degree. C.) are washed twice in the presence of sodium citrate
buffer, containing neither sucrose nor mannitol, before being taken
up in 50 mM Tris-HCl buffer (pH 7.2) enriched in NaCl (1M).
The alpha-amylase activity used to determine the eliciting
potential of compositions E1 and E2 is analyzed according to the
following protocol:
An enzymatic extract is prepared at +4.degree. C. from the elicited
or non-elicited cells. The cells, taken up in 50 mM Tris-HCl buffer
(pH 7.2) enriched in NaCl (1M) (1/5 v/v), are ground with a
Polytron homogenizer; the sample is kept in crushed ice throughout
the operation. The ground product is centrifuged (1200 g, 15 min at
4.degree. C.) and the supernatant is concentrated by
ultrafiltration on an Amicon cell equipped with a PM 10 membrane
(cut-off threshold 10,000 Daltons), and is then dialyzed against
distilled water with the aid of Ultrafrec units (Millipore)
(cut-off threshold 10,000 Da).
The alpha-amylase activity is assayed after incubation of the
enzymatic extract (10 .mu.g of proteins) at 40.degree. C. for a
variable time (1 to 6 hours) in the pre sence of the substrate (50
.mu.g of soluble starch) in 200 .mu.l of 0.1M sodium acetate buffer
(pH 5.0) or 0.1M sodium phosphate buffer (pH 7.0). The reaction is
stopped by heating at 100.degree. C. for 10 minutes.
The assay of the alpha-amylase activity is based on quantification
of the reducing sugars released, by Somogyi's method (1952). The
blanks consist of reaction media containing no enzymatic extract or
containing extracts heated to 100.degree. C.
The enzymatic activity (A) is expressed in .mu.mol of glucose
equivalents released per hour of enzymatic reaction and per mg of
proteins.
RESULTS
1. Expression of the Results
The enzymatic activation (R) is defined as the ratio of the
activity measured in elicited cells or protoplasts to the activity
measured in untreated cells or protoplasts, the activity of the
untreated cells or protoplasts being arbitrarily set at 100.
2. Results: Elicitation of Alpha-Amylase Activity
The results obtained have been plotted in FIG. 1.
Each value plotted in this Figure results from the processing of 3
experimental data obtained in the course of an elicitation
experiment.
Laminarin-based composition E1 induces an alpha-amylase activation
R of 300% in the cells, where as laminarin-based composition E2
induces an alpha-amylase activation R of 600%.
CONCLUSIONS
The results obtained show that the composition produced in Example
no. 2 and, to a lesser extent, that produced in Example no. 1 are
capable of stimulating an alpha-amylase activity, alpha-amylase
being a marker enzyme of the growth process, which is involved
especially during initial germination phases.
Laminarin can therefore be used as a seed germination and plant
growth accelerator.
Different compositions for agricultural use, containing laminarin
produced in Examples 1 and 2, will be given in Examples 5 to 8.
EXAMPLE 5
Composition for Agricultural use in the Form of Liquid Fertilizer
Based on Laminarin Produced in Example 1
The laminarin produced in Example 1 can be used in aqueous solution
with one or more deficiency-correcting trace elements, for example
a molybdenum salt and a manganese salt.
1 g of laminarin is dissolved in 758 g of water and then 215 g of
manganese chloride and 26 g of sodium molybdate are added. The
mixture is stirred until dissolution is complete.
This gives a composition having a laminarin content of 0.1%, a
molybdenum content of 1% by weight and a manganese content of 1% by
weight. This composition can be used as a plant growth stimulant
and deficiency corrector by spraying the leaves.
EXAMPLE 6
Agricultural Composition Based on Laminarin Produced in Example 2
in Association with a Fungicide
1 g of laminarin is dissolved in 699 g of water.
300 g of the fungicide Manganil 80 marketed by Bourgeois (no. APV
7000073), containing 80% of the active substance maneb, are
suspended in this solution.
This suspension, containing 0.1% of laminarin and 24% of maneb, can
be used by spraying on to the surface of the leaves in doses of
between 0.1 and 50 ml/l.
This composition exerts an accelerating action on plant growth at
the same time as fungicidal protection, in particular against vine
and tomato mildew.
EXAMPLE 7
Composition Based on Laminarin Produced in Example 1, used as a
Seed Coating Product
The laminarin produced in Example 1 can be used in aqueous solution
for film-coating a seed of the proteaginous pea type (Ascona
variety).
The pea seed, which may or may not have received a plant health
treatment, is film-coated with a film-coating machine of trademark
HEGE, which is based on the principle of spraying the substances at
very high speed on to a mass of seeds rotating about an axis; this
causes an eccentric displacement, ensuring perfect intimacy between
the seed and the film-coating products.
This method is used to prepare a batch of 500 g of pea seeds,
film-coated with 4 ml of an aqueous solution of laminarin produced
in Example no. 1, having a content of 5% and completed with a
dispersant for improving the product/seed contact.
This composition for film-coating seeds thus has an accelerating
action on seed germination and plantlet growth.
EXAMPLE 8
Composition Based on Laminarin Produced in Example 2, employed in
the Preparation of Tablets for Agricultural Use
Laminarin in powder form, produced in Example no. 2, can be used in
the preparation of tablets for watering indoor plants or spraying
their leaves.
0.5 g of laminarin produced in Example no. 2 is ground and mixed in
a mortar with 0.5 g of a dehydrating agent of the kaolin type. The
powder obtained is then compressed in a tableting machine of the
alternating type, comprising one fixed die and two punches (one
upper and one lower) moving in the vertical direction.
The 1 g granule or tablet obtained is then ejected from the machine
and can be used by dissolution in water in the watering can or
sprayer.
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